Prenatal Arrays user guide
Microarray Comparative Genomic Hybridisation (array CGH) has been in service
in the Leeds Cytogenetics lab since 2012 as a first line postnatal investigation for
children with unexplained learning difficulties and/or dysmorphic features. The
technology provides a high resolution genome-wide analysis of genomic
imbalance and as a result the abnormality detection rate is twice that achieved by
conventional cytogenetics.
As chromosomal anomalies account for 20~25% of major fetal anomalies, array
CGH is also increasingly being used to replace karyotyping for this referral group.
There have been several recent studies on the application of array CGH in
prenatal diagnosis which show that array CGH is more effective than karyotyping
at detecting pathogenic genomic imbalance [refs PD Brady et al, 2014, B Oneda
et al, 2014, and RJ Wapner et al, 2014, Vanakker et al 2014.
In the UK, an EME-funded study (the EACH project) which ran between 2012 and
2014 was designed to evaluate the patient and professional views of the
technology and the barriers/facilitators to its implementation into service. This
study ended in May 2014 and is due to report the findings. The Leeds
Cytogenetics lab participated in this study and has capitalised on its involvement
to develop robust and reliable processes and testing pathways for prenatal
arrays. It is anticipated that there will be a recommendation that prenatal arrays
are introduced into diagnostic service for referrals with abnormal ultrasound
findings. In the meantime, clear local guidelines have been produced detailing
how the Leeds cytogenetic lab applies array CGH in a prenatal setting.
This document is designed to provide current information about the local policy
regarding prenatal arrays and information about the technology. The local policy
is subject to amendment following publications and recommendations from the
EACH study, and any recommendations set by the Royal College of
Obstetricians and Gynaecologists and the Association for Clinical Genetic
Science (ACGS) Quality Committee (for best practice guidelines). Users will be
informed of any major changes in policy.
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What is array CGH and what are the benefits
Figure of chromosome 8 showing a loss of chromosomal material denoted by
the red bar
Array CGH is a technique for detecting abnormalities of genomic copy number. It is a
high resolution whole genome screen which detects genetic imbalances at a greater
resolution (~60Kb) compared with karyotyping (5-10Mb), ie a ~1000 fold increase in
resolution. This increase in resolution detects a higher number of genomic gains and
losses (imbalances) than can be seen using QF-PCR & karyotype. Array CGH also
accurately identifies the pathogenic genes involved in chromosome imbalances. This
gives more accurate information on the cause of the fetus’s phenotype and enables
better patient management.
Who will be tested by array CGH?
Prenatal array CGH will be introduced into our laboratory for selected cases for all
referring centers on the 13th February 2015. All referrals will initially be tested by
QF-PCR for sexing and rapid aneuploidy assessment for chromosomes X,Y,13,18
and 21. Where the QF-PCR result is normal prenatal arrays will be performed in the
following cases unless stated on the referral form by the referring clinician:
1)
2)
3)
4)
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A pregnancy with an abnormal ultrasound scan
A pregnancy with an increased NT of >3.5mm
Other specific clinician requests
A pregnancy in a family known to carry a clinically significant genomic
imbalance detected pre- or postnatally by array CGH or another molecular
method.
Specimens required for QF-PCR, Array CGH and karyotype to be performed:
Amniotic Fluid - Minimum 15-20ml
CVS - 5 fronds
Parental bloods will be requested by the laboratory if required
Suboptimal Samples
Where DNA quality from uncultured tissue is unsuitable for array CGH, it may be
necessary to extract DNA from cultured cells. In such circumstances, the lab will
notify the referring obstetrician of the need for performing array CGH on cultured
cells as this will generate a delay in reporting time.
Prenatal Pathway
AF or CVS
Serum screen risk
or maternal age
>3.5mm nuchal or
other ab scan
FH balanced
chromosome
abnormality
QF-PCR
13,18,21,X,Y
QF-PCR
13,18,21,X,Y
QF-PCR
13,18,21,X,Y
Normal Result
Abnormal Result
Normal Result
Report
Report
Report
Report
Array CGH
Culture
Report
Report
Culture
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Report
Normal Result
Abnormal Result
Turnaround Times (TAT)
Our professional body the Association for Clinical Genetic Science (ACGS) issues
best practice guidelines which define the recommended TAT for prenatal karyotyping
as 95% of cases reported within 14 calendar days of sample receipt. At present it is
not feasible to turn prenatal arrays around within this timeframe, although all efforts
will be made by the lab to work towards achieving this.
The lab will initially endeavour to report a prenatal array result within 21 days of
sample receipt for a first line array, or within 28 days for an array performed after
karyotyping due to an abnormal result. This will include any confirmatory studies
necessary in the event of finding a pathogenic imbalance.
Where it is necessary to analyse parental samples for confirmation/clarification
purposes, an interim prenatal array CGH report will be issued and a final report when
any necessary parental studies are completed.
Where DNA quality from uncultured tissue is unsuitable for array CGH, it may be
necessary to extract DNA from cultured cells. This will add on further delays to
reporting times as DNA extraction can only take place when cultures are ready
(about 14 days from receipt).
Platform used
Array CGH will be performed on extracted DNA using 8x60k ISCA design
oligonucleotide arrays supplied through Illumina. The average resolution of the array
is 60Kb. Only clinically significant imbalances will be reported.
Interpretation and Reporting
The purpose of a prenatal array in an ongoing pregnancy is to ascertain whether
there is a genetic imbalance present that is clinically significant with respect to the
features detected on antenatal scan or whether a familial imbalance is present in a
fetus.
Interpretation for ongoing pregnancies
1) Imbalances will only be reported if they are interpreted as likely to be clinically
significant to the fetus in relation to the supplied USS features. Guidance will be
taken from the locally held table produced by the EACH study. The guidelines were
devised with multicenter input. Clinically significant findings will include recognised
microdeletion and duplication syndromes, as well as other imbalances where the
literature details association with significant problems.
2) Neurosusceptibility loci have variable expression and penetrance and therefore
the phenotype of an individual with one of these imbalances may vary from within
normal limits to developmental delay, autism and schizophrenia. As phenotypic
certainty cannot be offered and in line with national consensus only a limited number
of these loci that are associated with an increased incidence of anomalies detected
on scan will be reported.
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3) Due to the increased resolution of the screening there may be occasional
unexpected incidental findings. Incidental findings will be discussed with the
obstetrician, Clinical Geneticists and laboratory staff on a case to case basis and will
only be reported if they will have implications later for the individual or relative and by
reporting them there will be a health benefit.
4) Benign imbalances or imbalances classified as variants of uncertain significance
(either likely benign or uncertain) will not be reported and will not be validated.
Interpretation for non-viable / terminated pregnancies
If a pregnancy is no longer ongoing (after discussion with obstetric team/clinical
geneticist) and on analysis an imbalance is detected that may either:
1) Have contributed to the phenotypic features of the fetus or the demise
of the pregnancy, or
2) Be clinically significant for other family members (neurosusceptibility
loci, incidental findings).
These findings will be reported.
Confirmation and follow-up
If a clinically significant imbalance is detected further confirmation studies will be
performed. These may take the form of FISH studies, Q-PCR (different to QF-PCR)
studies, MLPA, karyotyping or other techniques as appropriate.
Parental studies will also be undertaken to determine whether the imbalance has
arisen de-novo or has been inherited.
These studies may add extra days to the reporting time.
Reports
Following interpretation of the imbalances and adhering to the policy detailed above
reports will take the following format:
1) No imbalances detected relevant to the fetus’s problems.
NO CLINICALLY SIGNIFICANT IMBALANCE DETECTED
Microarray analysis on DNA extracted from chorionic villi/amniotic fluid sample has shown a fe/male
profile with no imbalances of known clinical significance detected. As such no further work will be
carried out on this sample.
Technical details: BlueGnome ISCA 8x60k OligoArray v2.0 array (chip number ******) and analysed with
Bluefuse Multi software (v4.1, v2.0 gal, ensembl build 70, GRCh37).
2) Imbalance detected which can be associated with the scan findings Abnormal Report
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FINAL REPORT: FAMILIALLY INHERITED 5p DELETION AND 16p DUPLICATION DETECTED BY ARRAYCGH AND CONFIRMED BY G-BANDING
Microarray analysis of DNA extracted from direct chronic villus material has shown a female profile with
two large imbalances involving material derived from the short arm of chromosome 5 and the short arm
of a chromosome 16. The first is a deletion of 5p15.33-5p15.33 which is estimated to be between a
minimum of 2.1Mb (DNA positions:22179-2167668) and a maximum of 2.3Mb (DNA positions:1-2274717.5)
in size and contains 26 genes including 5 OMIM morbid genes.
The second imbalance detected is a duplication of 16p13.3-16p11.2 which is estimated to be 32Mb
(minimum DNA positions:93748-32066997 and maximum DNA positions:1-32091684.5) in size. This
region contains 428 genes including 55 OMIM morbid genes. This patient has been added to decipher
showing the genes included in this region (see attached Decipher report - YRxxxxx).
G-banded chromosome analysis has confirmed that these imbalances are the result of a maternally
derived unbalanced translocation between the short arm of a chromosome 5 and the short arm of a
chromosome 16, with the duplicated 16 material positioned on the deleted 5p (see CVxxxx and Bxxxx). It
is highly likely that this unbalanced rearrangement is the cause of the scan findings seen in this foetus.
Advice on prenatal diagnosis may be helpful in any future pregnancy involving xxx and her partner. This
patient and her partner may benefit from genetic counselling which can be arranged at the Department of
Clinical Genetics, Chapel Allerton, Leeds.
Technical details: BlueGnome ISCA 8x60k OligoArray v2.0 array (chip number ******) and analysed with
Bluefuse Multi software (v3.1, v2.0 gal, ensembl build 62, GRCh37).
Results
All array results will be telephoned to the obstetricians. Unusual abnormal array
results will also be given to a Clinical Geneticist.
Limits to Array CGH technique and reporting restrictions
- Array CGH will not detect all genetic abnormalities
- Array CGH cannot detect balanced chromosome rearrangements such as
reciprocal translocations and inversions. Karyotyping will be carried out for patients
suspected of these abnormalities.
- Array CGH cannot detect low level mosaicism (< 30% mixture of abnormal and
normal cells)
- Anomalies may occur which cannot always be recognised or properly interpreted.
- Not all imbalances will be reported (as detailed above and in the laboratories policy
on prenatal arrays). Some unreported imbalances may become clinically significant
postnatally.
Frequently Asked Questions
What if I can’t obtain parental blood samples?
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We are aware that family circumstances can be difficult and it may not be possible to
obtain parental samples in some cases. Interpretation of an imbalance detected on
array CGH is less informative without inheritance studies.
Will array CGH detect mutations?
No. The array platform we will be using will detect small genomic imbalances which
may disrupt genes and give a similar phenotype to a mutation but it is not sensitive
enough to detect mutations within genes. Requests for specific mutation testing can
be made to the Leeds DNA laboratory.
Which referral card do I use?
It's the Cytogenetics and DNA Joint referral card which you already use.
It is important that any patient referred for array CGH testing is appropriately
consented and counselled about the technology, its limitations, target turnaround
times, and reporting arrangements.
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